CN110372790A - A kind of screening technique of across hoof-and-mouth disease serotypes monoclonal antibody - Google Patents
A kind of screening technique of across hoof-and-mouth disease serotypes monoclonal antibody Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The present invention relates to a kind of screening techniques of across hoof-and-mouth disease serotypes monoclonal antibody, belong to monoclonal antibody screening technique field.The present invention marks different serotype antigens using different fluoresceins, the specific b cells for obtaining and combining not synantigen are directly sorted by polychrome flow cytometer, designing that one kind is quickly obtained can be with the antigen reactive monoclonal antibody screening technique of different serotypes, it can obtain quickly generating across serum monoclonal antibody, obtaining the type monoclonal antibody can be used for identifying conservative antigen site between different virus serotype, can establish premise essential tool for serum dependent/non-dependent ELISA detection method.
Description
Technical field
The present invention relates to monoclonal antibody screening technique fields, and in particular to a kind of across hoof-and-mouth disease serotypes monoclonal
The screening technique of antibody, the i.e. screening technique of A type and O-shaped foot and mouth disease virus cross reaction monoclonal antibody.
Background technique
Aftosa (foot-and-mouth disease, FMD) is the great animal epidemic for seriously endangering pig, Niu Yuyang,
Massive losses are caused to China's animal husbandry.Foot and mouth disease virus (FMDV) has seven serotypes, respectively O-shaped, A type,
1 type of Asia, c-type, the type of SAT1~3, wherein each serotype again according to popular region division be multiple topological types (VP1,
85% amino acid identity).China's Major Epidemic is O-shaped and A type FMDV, wherein O-shaped FMDV is in China, mainly there are three topological types:
Southeast hypotype (SEA), Chinese classical type (Cathay) and the Middle East-South Asia type (ME-SEA)；A type FMDV is primarily present Asia topology
G1 and G2 branch in type.Different topology in Cross immunogenicity or even same serotype cannot be provided between different serotypes
There is also the differences of antigenic structure between type virus, cannot provide good Cross immunogenicity to heterologous strain.It can be seen that
FMDV serotype is numerous, although can prepare good vaccine strain by Reverse Genetics, is directed to different serotypes
Between the research of vaccine strain with cross-protection be still a huge difficult problem because being protected between determining FMDV serotype
Antigenic structure is kept clearly to recognize not yet.Parsing across conservative antigen structure between serotype is antiviral immunity research always
A focus, although thering is a large amount of research to report in FMDV serotype endoantigen site, between FMDV serotype
Conservative antigenic structure research still has blank.
Summary of the invention
The purpose of the present invention is to provide a kind of screening techniques of across hoof-and-mouth disease serotypes monoclonal antibody.Screening side
Method has great importance to the identification of conservative antigen structure FMDV serotype.It can be broad-spectrum long-acting aftosa molecular vaccine
Development lay the foundation, provide precondition to establish FMDV serotype dependent/non-dependent ELISA detection method.
The present invention provides a kind of screening techniques of across hoof-and-mouth disease serotypes monoclonal antibody, comprising the following steps:
1) separating peripheral blood mononuclear cells from the animal blood sample that O-shaped and A type FMDV bivalent inactivated vaccine is immunized；
2) the first ultrafiltration centrifugation is carried out to O-shaped FMDV 146S antigen, the O-shaped FMDV 146S antigen after being concentrated makes
The O-shaped FMDV 146S antigen after concentration is dyed with the first dyestuff, the second ultrafiltration centrifugation, the O-shaped FMDV after being marked
146S antigen；
3) third ultrafiltration centrifugation is carried out to A type FMDV 146S antigen, the A type FMDV 146S antigen after being concentrated makes
The A type FMDV 146S antigen after concentration is dyed with the second dyestuff, the 4th ultrafiltration centrifugation, the A type FMDV after being marked
146S antigen；
4) the O-shaped FMDV 146S after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains is anti-
A type FMDV 146S antigen, the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence of mouse after the label that former, step 3) obtains is anti-
Body mixing, the cell after being dyed；
It 5) can be in combination with O-shaped and A type FMDV double positive single B cells using polychrome selected by flow cytometry apoptosis；
6) reverse transcription is carried out to double positive single B cells that step 5) obtains, is expanded, is obtained single after obtaining cDNA
The heavy chain variable region gene and light-chain variable region gene of B cell IgG antibody；
7) the framework region front end of the heavy chain variable region gene for the single B cell IgG antibody for obtaining step 6) introduces such as SEQ
It is excellent to carry out gene order with reference to Homo sapiens (Human) codon for first secretion signal peptide sequence shown in ID NO.9
Change, the gene after optimization is inserted into the pcDNA3.4 carrier containing IgG heavy chain constant region, obtains heavy chain plasmid；By step 6)
The framework region front end of the light-chain variable region gene of obtained single B cell IgG antibody introduces as shown in SEQ ID NO.10 the
Two secretion signal peptide sequences carry out gene order optimization with reference to Homo sapiens (Human) codon, by the gene after optimization
It is inserted into the pcDNA3.4 carrier containing IgG constant region of light chain, obtains light chain plasmids；
8) heavy chain plasmid and light chain plasmids the transfection cell obtained step 7) carries out the expression of antibody；
And 3) the not restriction of chronological order the step 1), 2).
Preferably, step 2) first dyestuff include FluoProbes 647H, Allophycocyanin,
DyLight 633, DyLight 650, Atto 633 or Cynaine Dye 5.
Preferably, step 3) second dyestuff includes Pacific BlueTM, DyLight 405 or Atto390.
Preferably, step 2) the first ultrafiltration centrifugation, the second ultrafiltration centrifugation or step 3) the third ultrafiltration centrifugation
It independently is: 10~30min is centrifuged using 2500~4000rpm of super filter tube of 50~300kDa of interception, by antigen or label
Antigen afterwards is replaced into PBS buffer solution.
Preferably, step 3) the 4th ultrafiltration centrifugation are as follows: after being diluted with PBS buffer solution, using interception 50~
2500~4000rpm of super filter tube of 300kDa is centrifuged 10~30min, discards and flows through liquid, add repeatedly PBS buffer solution three times into
Row ultrafiltration centrifugal concentrating.
Preferably, the step 5) sorting are as follows: the 96-PCR orifice plate that 10 μ L lysates are contained in every hole is put into sorting cabin,
Every hole is set and sorts 1 cell pattern, loading；It draws door and irises out lymph and monocyte, excluded according to FSC-A and FSA-H setting
Adhesion cells iris out diagonal line single cell；To CD21+IgM-O-FMDV+A-FMDV+Cell carries out shooting sorting.
Preferably, the step 6) amplification includes nested PCR amplification method.
Preferably, the step 8) cell includes Expi293 suspension cell.
Preferably, the step 8) heavy chain plasmid and the molar ratio of light chain plasmids mixing are 1:(1~3).
The present invention provides a kind of screening techniques of across hoof-and-mouth disease serotypes monoclonal antibody.The present invention utilizes difference
Fluorescein marks different serotype antigens, and the specificity for obtaining and combining not synantigen is directly sorted by polychrome flow cytometer
B cell, designing that one kind is quickly obtained can be with the antigen reactive monoclonal antibody screening technique of different serotypes, and can obtain can
Quickly to generate across serum monoclonal antibody, obtains between the type monoclonal antibody can be used for identifying different virus serotype and guard
Antigen site can establish premise essential tool for serum dependent/non-dependent ELISA detection method.In embodiments of the present invention,
With hoof-and-mouth disease (foot-and-mouth disease virus, FMDV) for model antigen, successfully obtain can be with O, A type for screening
The monoclonal antibody of FMDV reaction, screening technique of the present invention have weight to the identification of conservative antigen structure FMDV serotype
Directive significance is wanted, can be laid the foundation for the development of broad-spectrum long-acting aftosa molecular vaccine, to establish FMDV serotype dependent/non-dependent
ELISA detection method provides precondition.
Detailed description of the invention
Fig. 1 is O, A type FMDV antigen Electronic Speculum observation (25000 times of amplifications) of fluorochrome label；
Fig. 2 is the bis- single B cells of the positive of airflow classification O, A type FMDV；
Fig. 3 is IgG antibody variable region gene PCR amplification；
Fig. 4 is expression and IgG antibody after purification；
Fig. 5 is IFA testing result；
Fig. 6 is indirect ELISA experimental result；
Fig. 7 is WB experimental result.
Specific embodiment
The present invention provides a kind of screening techniques of across hoof-and-mouth disease serotypes monoclonal antibody, comprising the following steps:
1) separating peripheral blood mononuclear cells from the animal blood sample that O-shaped and A type FMDV bivalent inactivated vaccine is immunized；
2) the first ultrafiltration centrifugation is carried out to O-shaped FMDV 146S antigen, the O-shaped FMDV 146S antigen after being concentrated makes
The O-shaped FMDV 146S antigen after concentration is dyed with the first dyestuff, the second ultrafiltration centrifugation, the O-shaped FMDV after being marked
146S antigen；
3) third ultrafiltration centrifugation is carried out to A type FMDV 146S antigen, the A type FMDV 146S antigen after being concentrated makes
The A type FMDV 146S antigen after concentration is dyed with the second dyestuff, the 4th ultrafiltration centrifugation, the A type FMDV after being marked
146S antigen；
4) the O-shaped FMDV 146S after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains is anti-
A type FMDV 146S antigen, the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence of mouse after the label that former, step 3) obtains is anti-
Body mixing, the cell after being dyed；
It 5) can be in combination with O-shaped and A type FMDV double positive single B cells using polychrome selected by flow cytometry apoptosis；
6) reverse transcription is carried out to double positive single B cells that step 5) obtains, is expanded, is obtained single after obtaining cDNA
The heavy chain variable region gene and light-chain variable region gene of B cell IgG antibody；
7) the framework region front end of the heavy chain variable region gene for the single B cell IgG antibody for obtaining step 6) introduces such as SEQ
It is excellent to carry out gene order with reference to Homo sapiens (Human) codon for first secretion signal peptide sequence shown in ID NO.9
Change, the gene after optimization is inserted into the pcDNA3.4 carrier containing IgG heavy chain constant region, obtains heavy chain plasmid；By step 6)
The framework region front end of the light-chain variable region gene of obtained single B cell IgG antibody introduces as shown in SEQ ID NO.10 the
Two secretion signal peptide sequences carry out gene order optimization with reference to Homo sapiens (Human) codon, by the gene after optimization
It is inserted into the pcDNA3.4 carrier containing IgG constant region of light chain, obtains light chain plasmids；
8) heavy chain plasmid and light chain plasmids the transfection cell obtained step 7) carries out the expression of antibody；
And 3) the not restriction of chronological order the step 1), 2).
The present invention separates the single core of peripheral blood from the animal blood sample that O-shaped and A type FMDV bivalent inactivated vaccine is immunized
Cell.The present invention is not particularly limited the immune method, using routine immunization method well known to those skilled in the art
?.The present invention is not particularly limited the type of the animal.In embodiments of the present invention, the animal is preferably ox.When
When the animal is preferably ox, the present invention preferably takes immune O, A type FMDV bivalent vaccine 14~30 days, and more preferably 21 days
Ox peripheral blood is as blood sample.
The present invention carries out the first ultrafiltration centrifugation to O-shaped FMDV 146S antigen, and the O-shaped FMDV 146S after being concentrated is anti-
Original dyes the O-shaped FMDV 146S antigen after concentration using the first dyestuff, the second ultrafiltration centrifugation, the O after being marked
Type FMDV 146S antigen.In the present invention, first dyestuff include FluoProbes 647H, Allophycocyanin,
DyLight 633, DyLight 650, Atto 633 or Cynaine Dye 5.The present invention to FluoProbes 647H,
Allophycocyanin, DyLight 633, DyLight 650, the source of Atto 633 or Cynaine Dye 5 be not special
It limits, using above-mentioned dyestuff conventional commercial product, such as Lightning-LinkTMRapidFluoProbes 647H label
Kit (InnovaBiosciences, San Diego, USA), Lightning-LinkTMAllophycocyanin label examination
Agent box (Innova Biosciences, San Diego, USA), Lightning-LinkTM633 labelling kit of DyLight
(InnovaBiosciences, San Diego, USA), Lightning-LinkTM650 labelling kit of DyLight
(Innova Biosciences, San Diego, USA), Lightning-LinkTM633 labelling kit (Innova of Atto
Biosciences, San Diego, USA) or Lightning-LinkTM5 labelling kit of Cynaine Dye
(InnovaBiosciences, San Diego, USA).In the present invention, interception 50 is preferably used in the first ultrafiltration centrifugation
~300kDa, the more preferably super filter tube of 100kDa replace O-shaped FMDV 146S antigen into PBS buffer solution, 2500~
4000rpm is centrifuged 10~30min, and more preferable 4000rpm is centrifuged 10min, concentrated antigen to 1mg/ml.The present invention is to the dyeing
Process be not particularly limited, according to above-mentioned dye reagent box carry out routine operation.In the present invention, second ultrafiltration
50~300kDa is preferably used in centrifugation, and the super filter tube of more preferably 100kDa is centrifuged, 2500~4000rpm, 10~30min of centrifugation,
More preferable 4000rpm is centrifuged 10min, and the antigen after label is replaced into PBS buffer solution.O-shaped FMDV after being marked
After 146S antigen, the present invention is preferably added to isometric 100% glycerol, is saved in -70 DEG C.
The present invention carries out third ultrafiltration centrifugation to A type FMDV 146S antigen, and the A type FMDV 146S after being concentrated is anti-
Original dyes the A type FMDV 146S antigen after concentration using the second dyestuff, the 4th ultrafiltration centrifugation, the A after being marked
Type FMDV 146S antigen.In the present invention, second dyestuff includes Pacific BlueTM, DyLight405 or Atto390.
The present invention is to the Pacific BlueTM, DyLight405 or Atto390 source be not particularly limited, using above-mentioned dyestuff
Conventional commercial product, such as Pacific BlueTMAntibody labeling kit (Thermo Scientific, USA),
Lightning-LinkTMDyLight405 labelling kit (InnovaBiosciences, San Diego, USA) and
Lightning-LinkTMAtto390 labelling kit (Innova Biosciences, San Diego, USA).In the present invention
In, A type FMDV 146S antigenic mark is not preferably interfered with each other between O-shaped FMDV 146S labelled antigen dyestuff with dyestuff, and two
Without adjusting fluorescence compensation between channel used in person.In the present invention, third ultrafiltration centrifugation preferably with interception 50~
300kDa, more preferably 100kDa super filter tube replace A type FMDV 146S antigen into PBS buffer solution, and adjustment antigen concentration is extremely
1mg/ml.The present invention is not particularly limited the process of the dyeing, carries out routine operation according to above-mentioned dye reagent box.
In the present invention, the product that the 4th ultrafiltration centrifugation is preferably drawn after dyeing is diluted with 10ml PBS buffer solution, is transferred to
50~300kDa, more preferably 100kDa super filter tube, 2500~4000rpm are centrifuged 10~30min, more preferable 4000rpm centrifugation
10min, discards and flows through liquid, adds 10ml PBS repeatedly three times, and concentration is preferably concentrated into 100 μ L.A type after being marked
After FMDV 146S antigen, the present invention is preferably added to isometric 100% glycerol, -70 DEG C of preservations.
After antigen after obtaining above two label, the present invention preferably takes staining method for electron microscopy to carry out the antigen after label
Observation, confirms whether the integrality of antigen particles is destroyed.It is verified, after the label that colouring method of the present invention obtains
There is no destroyed the integrality of A type FMDV 146S antigen after O-shaped FMDV 146S antigen and label.
The present invention by peripheral blood mononuclear cells and label after O-shaped FMDV 146S antigen, the A type FMDV after label
The anti-ox CD21-RPE of 146S antigen, mouse and the anti-ox IgM-FITC fluorescence antibody mixing of mouse, the cell after being dyed.In the present invention
In, the fluorescent antigen preferably includes the O-shaped FMDV 146S antigen and Pacific Blue after FluoProbes 647H label
A type FMDV 146S antigen after label.
The present invention can be in combination with O-shaped and A type FMDV double positive single B cells using polychrome selected by flow cytometry apoptosis.
The present invention is not particularly limited the model of the polychrome flow cytometer, and present invention preferably uses II u of BD FACSAria streams
Formula sorter, when using II u flow sorter of BD FACSAria, preferably instrument parameter is arranged by the present invention are as follows: nozzle is big
It is small: 100 μm；Sorting mode: single cell mode；Separation velocity: 10000 cells/second；Amplitude: 20psi；Oscillation frequency: 30kHz.
The above parameter setting closes Sweat button after adjusting, and using Accudrop, (article No.: 642412) delay microballoon, which executes, calculates liquid
Delay time.In the present invention, the sorting is preferred are as follows: the 96-PCR orifice plate that 10 μ L lysates are contained in every hole is put into sorting
Cabin is arranged every hole and sorts 1 cell pattern, loading；It draws door and irises out lymph and monocyte, arranged according to FSC-A and FSA-H setting
Except adhesion cells, diagonal line single cell is irised out；To CD21+IgM-O-FMDV+A-FMDV+Cell carries out shooting sorting.In this hair
In bright, before sorting, the hole 96-PCR Board position in sorting cabin is preferably adjusted, (is led to until sorting cell accurately falls into plate hole centre
It crosses using 96 orifice plates for being stained with sealed membrane, 60 cell patterns of every hole sorting is set and are adjusted, it is ensured that cell can be sorted into often
Hole center)
The present invention carries out reverse transcription to double positive single B cells, is expanded after obtaining cDNA, obtains single B cell IgG
The heavy chain variable region gene and light-chain variable region gene of antibody.The present invention is not particularly limited the reverse transcription method, uses
Conventional reverse transcription method.In the present invention, the amplification includes nested PCR amplification method.When animal of the present invention is
Niu Shi, present invention preferably employs nucleotide sequence primers as shown in NO.1~8 SEQ ID to be expanded.
The framework region front end of the heavy chain variable region gene of single B cell IgG antibody is introduced such as SEQ ID NO.9 by the present invention
Shown in the first secretion signal peptide sequence, with reference to Homo sapiens (Human) codon carry out gene order optimization, will optimize
VH gene afterwards is inserted into the pcDNA3.4 carrier containing IgG heavy chain constant region, obtains heavy chain plasmid；The list that step 6) is obtained
The framework region front end of the light-chain variable region gene of a B cell IgG antibody introduces the second secretion letter as shown in SEQ ID NO.10
Number peptide sequence carries out gene order optimization with reference to Homo sapiens (Human) codon, the VL gene after optimization is inserted into
PcDNA3.4 carrier containing IgG constant region of light chain, obtains light chain plasmids.
Transfection cell carries out the expression of antibody after the present invention mixes heavy chain plasmid and light chain plasmids.In the present invention, institute
Stating cell includes Expi293 suspension cell.In the present invention, the heavy chain plasmid and the molar ratio of light chain plasmids mixing are
1:(1~3).Specifically, the present invention expresses antibody by transiently transfecting Expi293 suspension cell, takes heavy chain plasmid and light chain matter
Grain is mixed, and OptiPRO is addedTMSFM culture medium is diluted, while taking equivalent OptiPROTMThe dilution of SFM culture medium
ExpiFectamineTM293 transfection reagents then mix the plasmid diluted and transfection reagent, and room temperature acts on 5min.
Combined with specific embodiments below to a kind of sieve of across hoof-and-mouth disease serotypes monoclonal antibody of the present invention
Choosing method is further described in detail, and technical solution of the present invention includes but is not limited to following embodiment.
Embodiment 1
1.FMDV antigenic mark scheme
1.1O type FMDV 146S antigenic mark scheme
Use Lightning-LinkTMRapid FluoProbes 647H labelling kit (Innova
Biosciences, San Diego, USA) the O-shaped FMDV 146S antigen of label.First with interception 100kDa super filter tube O-shaped
FMDV 146S antigen is replaced into PBS buffer solution.4000rpm is centrifuged 10min, concentrated antigen to 1mg/ml (OD260=7.6 about
Equal to 1mg/ml).It takes 100 μ L146S antigens that 10 μ L LL-modifierreagent are added, mixes gently, then therefrom draw
100 μ L are added to a pipe Lightning-LinkTMMix reactive dye, gently aspirates and powder is resuspended, (20-25 DEG C) of room temperature work
With 3 hours.10 μ L LL-quencherreagent are added, act on 30 minutes, to terminate reaction.Then 100kDa super filter tube is used
Centrifugation, 4000rpm are centrifuged 10min, the antigen after label are replaced into PBS buffer solution.It is added isometric 100% glycerol, -70
DEG C save.Antigen after label is named as O-FMDV 146S-FluoProbes 647H.
1.2A type FMDV 146S antigenic mark scheme
To avoid what between O-shaped FMDV 146S labelled antigen fluorescence compensated from interfering with each other, Pacific Blue is selectedTM
Antibody labeling kit (Thermo Scientific, USA) marks A type FMDV146S antigen.It is super with interception 100kDa first
Chimney filter replaces A type FMDV 146S antigen into PBS buffer solution, adjustment antigen concentration to 1mg/ml (OD260=7.6 are approximately equal to
1mg/ml).It takes 10 μ L sodium bicarbonate buffer liquid (1M, pH=8.3) that 100 μ L146S antigens are added, mixes gently, then therefrom inhale
It takes 100 μ L to be added to a tube reaction dyestuff, turns upside down 10 times, with abundant dissolving dye.(20-25 DEG C) of room temperature is incubated for 1 hour,
Period turned upside down 3 times every 10-15 minutes.Then the product after drawing dyeing is diluted with 10ml PBS buffer solution, is transferred to
100kDa super filter tube, 4000rpm are centrifuged 10min, discard and flow through liquid, add 10ml PBS repeatedly three times, after concentration label
Antigen is to 100 μ L.Isometric 100% glycerol, -70 DEG C of preservations are added.Antigen after label is named as A-FMDV 146S-
Pacific Blue。
The dyeing of 1.3 Electronic Speculum
To confirm that above-mentioned fluorochrome label O, A type FMDV antigen particles is selected not cause brokenly antigen particles integrality
It is bad, therefore negative staining and transmission electron microscope observing are carried out to the antigen after label.4 microlitres of antigen after taking label are gently added copper mesh
On, room temperature acts on 2min, then laterally blots sample with filter paper.Then sample is clamped in 1% phosphoric acid tungsten dyeing liquor with tweezers
Shuttle dyeing is carried out, using 3 drop methods, every drop dye liquor acts on 10 seconds, then draws dye liquor with filter paper, dry, Electronic Speculum observation.
2. polychrome streaming Staining Protocol
2.1 peripheral blood mononuclear cells (PBMCs) separation
Take immune O, A type FMDV bivalent vaccine 21 days (- 30 days 14 days sections) ox peripheral blood, use lymphocyte point
Chaotropic (density=1.083g/mL) therefrom separating peripheral blood mononuclear cells (PBMCs), concrete operations are as follows:
(1) 6mL lymphocyte separation medium is added into 15mL centrifuge tube in lymphocyte separation medium and PBS equilibrium at room temperature.
(2) 1:1 dilution proportion ox EDTA anticoagulation is pressed with PBS, whole blood is added to separation of lymphocytes after drawing 8mL dilution
Liquid upper layer, 1200 × g are centrifuged 30min.
(3) milky-white layer peripheral blood mononuclear cells (PBMCs) cell is drawn to be added containing 1/2 volume cells sorting liquid
(contain 1%BSA, 2mM EDTANa2PBS) 15mL centrifuge tube in, 600 × g be centrifuged 5min.
(4) supernatant liquid is discarded, 1~2mL erythrocyte cracked liquid is added, cell sorting is added after 1~2min of lysis at room temperature
Liquid, 250 × g are centrifuged 10min.
(5) supernatant liquid is discarded, is washed twice of cell with cell sorting liquid, 400 × g is centrifuged 5min.Supernatant liquid is discarded, is added
Cell sorting liquid dispels as individual cells, and the cell finally obtained is peripheral blood mononuclear cells (PBMCs), counts.
The dyeing of 2.2 fluidic cells
(1) 10 are taken7The B cell of a enrichment is resuspended in 200 μ L cell sorting liquid, and 0.5 μ g O-FMDV 146S- is added
FluoProbes 647H antigen, 0.5 μ g A-FMDV 146S-Pacific Blue antigen, the 2 anti-ox CD21-RPE fluorescence of μ g mouse
Antibody (Bio-Rad, USA) and the anti-ox IgM-FITC fluorescence antibody (Bio-Rad, USA) of 1 μ g mouse, act on 25min on ice.Take phase
With cell setting Isotype control and the control that subtracts one.Dan Yangguan is set simultaneously, for adjusting compensation.
(2) cell sorting liquid wash cell primary, 400 × g, 4 DEG C of centrifugation 5min.
(3) cell is resuspended in 500 μ L cell sorting liquid, is protected from light on ice, machine sorts cell in preparation.
3. sorting the single B cell of O, A type FMDV cross reaction
Use the single B cell of II u flow sorter of BD FACSAria sorting FMDV specificity.Instrument setting parameter is to spray
Mouth size: 100 μm；Sorting mode: single cell mode；Separation velocity: 10000 cells/second；Amplitude: 20psi；Oscillation frequency:
30kHz.The above parameter setting closes Sweat button after adjusting, and using Accudrop, (article No.: 642412) delay microballoon executes meter
Calculate liquid delay time.Then the hole 96-PCR Board position in sorting cabin is adjusted, until sorting cell accurately falls into plate hole centre
(60 cell patterns of every hole sorting can be set and be adjusted by using 96 orifice plates for being stained with sealed membrane).Arrangement above is complete
The 96-PCR orifice plate that 10 μ L lysates are contained in every hole is put into sorting cabin, starts loading by Cheng Hou.It draws door and irises out lymph and monokaryon is thin
Born of the same parents exclude adhesion cells according to FSC-A and FSA-H setting, iris out diagonal line single cell.Then to CD21+IgM-O-FMDV+
A-FMDV+Cell carries out shooting sorting, can be obtained the single B cell of O, A type FMDV cross reaction.
4. single B cell source IgG antibody variable region gene amplification
4.1 unicellular cDNA molecule preparations
After the completion of sorting, 1 μ L terminate liquid is added in every hole, and room temperature acts on 5min, terminates reaction.Then 4 μ L are added in every hole
SuperScriopt VILO mix solution and 6 μ L DNase/RNase-free water are mixed.1500rpm, 4 DEG C of centrifugations
5min.PCR instrument carries out post transcription cloning.
Reaction condition such as table 1 is set
1 post transcription cloning condition of table
CDNA-20 DEG C of storage of acquisition, is used for subsequent PCR amplification.
The PCR amplification of 4.2 unicellular cDNA
Using primer shown in table 2, take nested PCR method to the heavy chain variable region (VH) of the single B cell IgG antibody of ox
Gene and lambda light chain variable region (VL) gene.
The primer of the amplification of table 2 ox IgG VH and VL gene
Degeneracy base annotation in sequence, S=C or G, Y=C or T, R=A or G.
First round PCR reaction system
The first round reaction system such as table 3 for expanding IgG VH and VL gene, in addition to primer is different with template, remaining ingredient is equal
It is carried out according to the system.
3 first round of table PCR reaction system
Because annealing temperature is different, response procedures involved in different primers are different, specific response procedures such as 4 He of table
Table 5:
4 IgG VL gene first round amplification program of table
5 IgG VH gene first round amplification program of table
Second wheel PCR reaction system
The second wheel reaction system of IgG VH and VL gene is expanded, and except (template is that first round PCR expands for primer and template
Increase production object) it is different, remaining ingredient is carried out according to system as shown in table 6:
Table 6 second takes turns PCR reaction system
The amplification program of second wheel PCR amplification IgG VH and VL gene is identical, and response procedures are as shown in table 7.
The amplification program of 7 IgG VH and VL gene of table
The sequencing of 4.3 PCR products
The PCR product for taking 5 μ L second wheel amplification carries out electrophoresis with 1.5% Ago-Gel, observes amplification.Success
Remaining 45 μ L product is carried out DNA sequencing, uses DNASTAR and SnapGene software by the sample for amplifying VH and VL gene
Analysis compares sequencing result.
5. prepared by vector construction and endotoxin-free plasmid
The framework region front end (FR1) of the IgG antibody VH gene of amplification is introduced into secretion signal peptide sequence
" MNPLWTLLFVLSAPRGVLS " (SEQ ID NO.9) carries out gene then referring to Homo sapiens (Human) codon
Sequence optimisation is inserted into the pcDNA3.4 carrier containing ox IgG heavy chain constant region by Not1 and Nhel restriction enzyme site.It will amplification
The VL gene framework region front end (FR1) introduce secretion signal peptide sequence " MAWSPLFLTLVAVYTGS " (SEQ ID NO.10), and
Gene order optimization is carried out with reference to Homo sapiens (Human) codon, is then inserted by Nhe1 and Ale1 restriction enzyme site
Onto the pcDNA3.4 carrier containing IgG constant region of light chain.The above antibody constant region gene/C terminal introduce 6 × His label and
Myc label, in favor of detecting and purifying.Sequent synthesis is synthesized with vector construction trust money Wei Zhi biotech company.Make
With a large amount of extraction agent boxes of endotoxin-free plasmid (TIANGEN, Beijing, China), in strict accordance with kit specification to building
Antibody expressing plasmid carries out endotoxin-free plasmid preparation.
6. the expression and purification of antibody
Antibody is expressed by transiently transfecting Expi293 suspension cell, takes 15 μ g of heavy chain plasmid and light chain matter in the ratio of 2:1
30 μ g of grain are mixed, and 250 μ L OptiPRO are addedTMSFM culture medium is diluted, while taking equivalent OptiPROTMSFM training
It supports base and dilutes ExpiFectamineTM293 transfection reagents then mix the plasmid diluted and transfection reagent, room temperature effect
5min.The mixture is slowly added to 1.5 × 108In a Expi293 cell.Expi293 cell is placed in 37 DEG C of perseverances after transfection
Warm suspension incubator suspends after culture 18h, adds 150 μ L Expi293TMEnhancer and 6mL Expi293TM Feed。37
DEG C continue after cultivating 9d, be centrifuged 30min by 10 000 × g, collect cells and supernatant, through 0.22 μm of filter filtering, after being used for
Continuous antibody purification.Antibody purification is carried out on AKAT protein purification instrument using HiTrap TALON pillar, uses 250mM imidazoles
PBS liquid antibody elution is then charged into bag filter and is placed in PBS liquid and dialyses 3 times, each 3h.Antibody after dialysis, with PEG 8
000 is concentrated, and SDS-PAGE electrophoretic analysis is carried out after concentration.
O, the antibody activity verifying of A type FMDV cross reaction
7.1 indirect immunofluorescences (indirect immunofluorescence assay, IFA) test
BHK-21 cell is accessed in 24 orifice plates, when cell grows to 80%, in the ratio of MOI=1 by O-shaped FMDV
(O/HN/CHA93 strain) and A type FMDV (A/GDMM/CHA/2013 strain) is inoculated with 24 porocyte culture plates respectively, is arranged simultaneously
Malicious normal cell controls are not connect collects supernatant inactivation treatment after being incubated for 4h in 37 DEG C of cell incubators.Then -20 DEG C are added in advance
The fixed cell of cold Jia Chun ︰ acetone (1:1) fixer, room temperature act on 20min.PBS is washed cell 3 times, is added by 5 μ g/mL concentration
The antibody of purifying, 37 DEG C of incubation 1h.PBS is washed 3 times, and the fluorescein label rabbit-anti ox IgG-FITC (Thermo of working concentration is added
Scientific, USA), 37 DEG C of incubation 30min.PBS is washed cell 5 times, and fluorescence microscope fluorescence signal simultaneously photographs to record.
7.2 western blots (WesternBlot, WB) test
Taking concentration respectively is the O-shaped FMDV (O/HN/CHA93 strain) and A type FMDV (A/GDMM/CHA/ of 0.1mg/mL
2013 strains) antigen carries out SDS-PAGE electrophoresis, isolated protein band is then transferred to cellulose nitrate (NC) film, with containing
The TBST buffer blind 2h of 5% skimmed milk power after washing, dilutes antibody to work with the TBST buffer containing 5% skimmed milk power
Make concentration (2 μ g/mL), be incubated at room temperature 2h, after TBST washes film, anti-His tag antibody (1 ︰ 4000) room temperature that HRP label is added is incubated
1h is educated, after TBST washes film, ECL chemiluminescent substrate is added, pressure X-ray is exposed imaging in darkroom.
7.2 indirect ELISA
O-shaped FMDV (O/HN/CHA93 strain) after purification is used with A type FMDV (A/GDMM/CHA/2013 strain) antigen
PBS is diluted to 1 μ g/mL, by 100 holes μ L/, 4 DEG C of overnight coated elisa plates；After PBST washes 5 times, 1% gelatin closes 1h, and PBST is washed
After 5 times, drying；Antibody to be checked is serially diluted for 2 times since 1 ︰ 100, is added in ELISA Plate by every 100 μ L of hole, 37 DEG C of incubation 1h；
PBST is washed 5 times, and anti-His tag antibody (1 ︰ 10000) 100 the μ l, 37 DEG C of incubation 1h of HRP label is added in every hole；PBST is washed 5 times,
100 μ L TMB developing solutions, color development at room temperature 10min is added；Terminate liquid color development stopping is added, with the suction at microplate reader measurement 450nm
Light value (D450).S/C.O mode counts, and S is sample D450Value, C.O, that is, Cut Off value (positive decision content), C.O=2.1 × N (N
For negative control D450Value, when N is less than 0.05 based on 0.05), S/C.O >=1 is determined as the positive, and S/C.O < 1 is determined as feminine gender.
Experimental result:
1, O, A type FMDV antigen particles integrality of fluorochrome label
After negative staining shown in Electronic Speculum observation result figure 1, wherein the O-shaped FMDV after Fig. 1-1 fluorescent marker；Fig. 1-2 fluorescent marker
A type FMDV afterwards, O-shaped FMDV and A type FMDV antigen particles 146S particle size after fluorochrome label is uniform, diameter 20-
30nm, shape are intact.Morphologic observation can guarantee the result shows that these dye marker antigens will not damage antigen particles
146 antigen particles integralities are consequently adapted to carry out sorting single B cell as bait antigen.
2, the bis- single B cells of the positive of O, A type FMDV are successfully obtained
Flow cytometer showed result is as shown in Figure 2, wherein A: machine in peripheral blood mononuclear cells (PBMCs) after dyeing irises out shape
The good P1 group of state；The shooting of B:P1 cell colony, irises out diagonal line single cell, excludes adhesion cells；C: CD21+IgM- is irised out
B cell；D: the FMO control of fluorescent antigen is not added；E: normal dyeing sample；Each cell colony ratio in F:FMO check sample
Cell (about 1,000,000 cells of record)；G: normal dyeing sample proportion (about 1,000,000 cells of record).The mono- positive B of A type FMDV
Cell (P4 group), the O-shaped mono- positive B-cells of FMDV (P5 group), the bis- positive B-cells of O, A type FMDV (P6 group) are high-visible
(Fig. 2 E), is present in CD21+IgM-Cell colony, the ratio of the total peripheral blood mononuclear cells of Zhan (PBMCs) respectively may be about 72/
1000000,91/1000000,19/1000000 (Fig. 2 F).Compared to subtracting one, control (FMO) sample (fluorescence target O, A type is not added
FMDV antigen), the mono- positive B-cells of A type FMDV (P4 group), the O-shaped mono- positive B-cells of FMDV (P5 group), the bis- sun of O, A type FMDV
Property B cell (P6 group) respectively may be about 4/1000000,1/1000000,0/1000000 (Fig. 2 G), illustrate be for O, A type FMDV
Is specifically bound accordingly by P6 group (CD21+IgM-O-FMDV+A-FMDV+B cell) circle door sorting, use unicellular point
Lectotype, successfully sorting obtains the single B cell of multiple bis- positives of O, A type FMDV.
The acquisition of ox IgG variable region gene in the bis- positive B-cells of 3.O, A type FMDV
It is as shown in Figure 3 that nested PCR product agarose gel electrophoresis analyzes result, wherein Fig. 3-1:IgG light chain variable region
PCR product electrophoresis；Fig. 3-2:IgG heavy chain variable region PCR product electrophoresis, IgG VL segment occur clearly between 450bp-500bp
Clear bands visible；There is clear band at 500bp-600bp in IgG VH segment.Sequencing result passes through Ig BLAST database ratio
It is right, it was demonstrated that VH the and VL segment that nested PCR amplification obtains is the IgG antibody variable region gene sequence of ox
4, successful expression and it is purified into IgG antibody molecule
SDS-PAGE result is as shown in Figure 4, wherein 1,3,5 swimming lanes are cell conditioned medium antibody-containing, and 2,4,6 swimming lanes are
Purify the IgG antibody obtained.The results show that expression antibody cell conditioned medium after affinitive layer purification, electrophoresis result and expection
Unanimously, show two high-visible bands at 60kDa and 30kDa respectively, respectively correspond the heavy chain of IgG antibody molecule and light
Chain.As a result confirm that this method successful expression and can be purified into IgG antibody molecule
5, O, A type FMDV cross reaction monoclonal antibody are successfully screened
5.1 IFA results
IFA testing result is as shown in Figure 5, wherein Fig. 5-1 is that monoclonal antibody 1 and O/HN/CHA93 virus strain infection BHK21 cell are real
It tests as a result, Fig. 5-2 is monoclonal antibody 1 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment as a result, Fig. 5-3 is full Niu Yuandan
Anti- 1 with normal BHK21 cell experiment as a result, Fig. 5-4 be monoclonal antibody 2 and O/HN/CHA93 virus strain infection BHK21 cell experiment as a result,
Fig. 5-5 be monoclonal antibody 2 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment as a result, Fig. 5-6 be full ox source monoclonal antibody 2 with just
Normal BHK21 cell experiment is as a result, Fig. 5-7 is monoclonal antibody 3 and O/HN/CHA93 virus strain infection BHK21 cell experiment as a result, Fig. 5-8 is
Monoclonal antibody 3 and A/GDMM/CHA/2013 virus strain infection BHK21 cell experiment are as a result, Fig. 5-9 is full ox source monoclonal antibody 3 and normal BHK21
Cell experiment result.The results show that 1,2,3 three strain antibodies can be with the BHK-21 cell and A/ of O/HN/CHA93 virus strain infection
There is specific green fluorescence in the BHK21 cell combination of GDMM/CHA/2013 virus strain infection, and do not connect the control cell of poison without
Visible green fluorescence.The result, which confirms that successfully screening obtains, to be resisted with the monoclonal of O-shaped FMDV and A type FMDV cross reaction
Body.
5.2 result of indirect ELISA
As a result as shown in Figure 6, wherein binding ability of Fig. 6-1 monoclonal antibody 1 to O-shaped FMDV and A type FMDV antigen；Fig. 6-2 is single
The binding ability of anti-2 pairs of O-shaped FMDV and A type FMDV antigen；Combination energy of Fig. 6-3 monoclonal antibody 3 to O-shaped FMDV and A type FMDV antigen
Power.Monoclonal antibody 1,2,3 can be with O-shaped and A type FMDV antigen binding, and three plants of monoclonal antibodies are to O-shaped and A type FMDV antigen relative affinity
Respectively≤0.01 μ g/mL ,≤0.01 μ g/mL ,≤0.01 μ g/mL, wherein monoclonal antibody 1 is higher than A type FMDV antigen affinity O-shaped
FMDV antigen, monoclonal antibody 2 and monoclonal antibody 3 are higher than A type FMDV antigen to O-shaped FMDV antigen affinity.
5.3WB result
Electrophoresis, WB result such as Fig. 7 institute are carried out to O/HN/CHA93 strain antigen and A/GDMM/CHA/2013 strain antigen
Show, wherein Fig. 7-1 is monoclonal antibody 1 and O/HN/CHA93 strain antigen and A/GDMM/CHA/2013 strain antigen-reactive result；Figure
7-2 is monoclonal antibody 2 and O/HN/CHA93 strain antigen and A/GDMM/CHA/2013 strain antigen-reactive result；Fig. 7-3 is monoclonal antibody 3
With O/HN/CHA93 strain antigen and A/GDMM/CHA/2013 strain antigen-reactive result.The results show that monoclonal antibody 1 25kDa with
Between 35kDa, occurs specific bond band at about 28kDa, corresponding FMDV Viral structural protein VP2, same monoclonal antibody 2 is also at this position
There is onesize band.And monoclonal antibody 3 is reactionless to O, A type FMDV antigen.The experimental result confirms that monoclonal antibody 1 and 2 can be same
When reacted with O-shaped FMDV and A type FMDV, and combine VP2 on linear epitope.Monoclonal antibody 3 may be in conjunction with the comformational epitope of FMDV.
IFA and ELISA experimental result confirms, screens 3 plants of monoclonal antibodies of acquisition to the equal energy of O-shaped FMDV and A type FMDV
It specifically binds.Further pass through WB it is experimentally confirmed that monoclonal antibody 1 and monoclonal antibody 2 identified is guarded on FMDV capsid protein VP2
Linear epitope, monoclonal antibody 3 identify O-shaped FMDV and A type FMDV between guard comformational epitope.Based on the above results, this research
The screening technique of offer can quickly screen across FMDV serotype monoclonal antibody, the conservative antigen between research FMDV serotype
Site provides good tool.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of screening technique of across hoof-and-mouth disease serotypes monoclonal antibody
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Claims (9)
1. a kind of screening technique of across hoof-and-mouth disease serotypes monoclonal antibody, comprising the following steps:
1) separating peripheral blood mononuclear cells from the animal blood sample that O-shaped and A type FMDV bivalent inactivated vaccine is immunized；
2) the first ultrafiltration centrifugation carried out to O-shaped FMDV 146S antigen, O-shaped FMDV 146S antigen after being concentrated uses the
One dyestuff dyes the O-shaped FMDV 146S antigen after concentration, the second ultrafiltration centrifugation, the O-shaped FMDV after being marked
146S antigen；
3) third ultrafiltration centrifugation carried out to A type FMDV 146S antigen, A type FMDV 146S antigen after being concentrated uses the
Two dyestuffs dye the A type FMDV 146S antigen after concentration, the 4th ultrafiltration centrifugation, the A type FMDV after being marked
146S antigen；
4) by the O-shaped FMDV 146S antigen after the peripheral blood mononuclear cells that step 1) obtains and the label that step 2) obtains, step
A type FMDV 146S antigen, the anti-ox CD21-RPE of mouse and the anti-ox IgM-FITC fluorescence antibody of mouse after the rapid label 3) obtained is mixed
It closes, the cell after being dyed；
It 5) can be in combination with O-shaped and A type FMDV double positive single B cells using polychrome selected by flow cytometry apoptosis；
6) reverse transcription is carried out to double positive single B cells that step 5) obtains, is expanded after obtaining cDNA, it is thin obtains single B
The heavy chain variable region gene and light-chain variable region gene of born of the same parents' IgG antibody；
7) the framework region front end of the heavy chain variable region gene for the single B cell IgG antibody for obtaining step 6) introduces such as SEQ ID
First secretion signal peptide sequence shown in NO.9 carries out gene order optimization with reference to Homo sapiens (Human) codon, will
Gene after optimization is inserted into the pcDNA3.4 carrier containing IgG heavy chain constant region, obtains heavy chain plasmid；
The framework region front end of the light-chain variable region gene for the single B cell IgG antibody that step 6) is obtained introduces such as SEQ ID
Second secretion signal peptide sequence shown in NO.10 carries out gene order optimization with reference to Homo sapiens (Human) codon,
Gene after optimization is inserted into the pcDNA3.4 carrier containing IgG constant region of light chain, obtains light chain plasmids；
8) heavy chain plasmid and light chain plasmids the transfection cell obtained step 7) carries out the expression of antibody；
And 3) the not restriction of chronological order the step 1), 2).
2. screening technique according to claim 1, which is characterized in that step 2) first dyestuff includes FluoProbes
647H, Allophycocyanin, DyLight 633, DyLight 650, Atto 633 or Cynaine Dye 5.
3. screening technique according to claim 1, which is characterized in that step 3) second dyestuff includes Pacific
BlueTM, DyLight 405 or Atto390.
4. screening technique according to claim 1, which is characterized in that step 2) the first ultrafiltration centrifugation, the second ultrafiltration
Centrifugation or step 3) third ultrafiltration centrifugation independently are: using 50~300kDa of interception super filter tube 2500~
4000rpm is centrifuged 10~30min, and the antigen after antigen or label is replaced into PBS buffer solution.
5. screening technique according to claim 1, which is characterized in that step 3) the 4th ultrafiltration centrifugation are as follows: slow with PBS
After fliud flushing dilution, it is centrifuged 10~30min using 2500~4000rpm of super filter tube of 50~300kDa of interception, discards and flows through liquid
Body adds PBS buffer solution repeatedly and carries out ultrafiltration centrifugal concentrating three times.
6. screening technique according to claim 1, which is characterized in that the step 5) sorting are as follows: 10 μ L are contained in every hole
The 96-PCR orifice plate of lysate is put into sorting cabin, and every hole is arranged and sorts 1 cell pattern, loading；It draws door and irises out lymph and monokaryon
Cell excludes adhesion cells according to FSC-A and FSA-H setting, irises out diagonal line single cell；To CD21+IgM-O-FMDV+A-
FMDV+Cell carries out shooting sorting.
7. screening technique according to claim 1, which is characterized in that the step 6) amplification includes nested PCR amplification side
Method.
8. screening technique according to claim 1, which is characterized in that the step 8) cell includes that Expi293 suspends carefully
Born of the same parents.
9. screening technique according to claim 1 or 8, which is characterized in that the step 8) heavy chain plasmid and the light chain
The molar ratio of plasmid mixing is 1:(1~3).
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